Multiplex Protein Profiling by Low-Signal-Loss Single-Cell Western Blotting with Fluorescent-Quenching Aptamers.
Li XuHaiyang XieBoqian WangZijian ZhuHui JiangXiaoqian DuanShuxin DengJiasu XuLai JiangXianting DingPublished in: Analytical chemistry (2023)
Single-cell western blotting (scWB) is a prevalent technique for high-resolution protein analysis on low-abundance cell samples. However, the extensive signal loss during repeated antibody stripping precludes multiplex protein detection. Herein, we introduce F luorescent-quenching A ptamer-based S ingle-cell W estern B lotting (FAS-WB) for multiplex protein detection at single-cell resolution. The minimal size of aptamer probes allows rapid in-gel penetration, diffusion, and elution. Meanwhile, the fluorophore-tagged aptamers, coordinated with complementary quenching strands, avoid the massive signal loss conventionally caused by antibody stripping during repeated staining. Such a strategy also facilitates multiplex protein analysis with a limited number of fluorescent tags. We demonstrated FAS-WB for co-imaging four biomarker proteins (EpCAM, PTK7, HER2, CA125) at single-cell resolution with lower signal loss and enhanced signal-to-noise ratio compared to conventional antibody-based scWB. Being more time-saving (less than 25 min per cycle) and economical (1/1000 cost of conventional antibody probes), FAS-WB offers a highly efficient platform for profiling multiplex proteins at single-cell resolution.
Keyphrases
- single cell
- high throughput
- rna seq
- real time pcr
- high resolution
- protein protein
- highly efficient
- single molecule
- label free
- living cells
- small molecule
- binding protein
- stem cells
- south africa
- gold nanoparticles
- fluorescence imaging
- mass spectrometry
- bone marrow
- nucleic acid
- antibiotic resistance genes
- liquid chromatography
- energy transfer